DIHYDROCHALCONES IN MALUS MILL. GERMPLASM AND HYBRID POPULATIONS A Dissertation Presented to the Faculty of the Graduate School of Cornell University In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy by Benjamin Leo Gutierrez December 2017 © 2017 Benjamin Leo Gutierrez DIHYDROCHALCONES IN MALUS MILL. GERMPLASM AND HYBRID POPULATIONS Benjamin Leo Gutierrez, Ph.D. Cornell University 2017 Dihydrochalcones are abundant in Malus Mill. species, including the cultivated apple (M. ×domestica Borkh.). Phloridzin, the primary dihydrochalcone in Malus species, has beneficial nutritional qualities, including antioxidant, anti-cancer, and anti-diabetic properties. As such, phloridzin could be a target for improvement of nutritional quality in new apple cultivars. In addition to phloridzin, a few rare Malus species produce trilobatin or sieboldin in place of phloridzin and hybridization can lead to combinations of phloridzin, trilobatin, or sieboldin in interspecific apple progenies. Trilobatin and sieboldin also have unique chemical properties that make them desirable targets for apple breeding, including high antioxidant activity, anti- inflammatory, anti-diabetic properties, and a high sweetness intensity. We studied the variation of phloridzin, sieboldin, and trilobatin content in leaves of 377 accessions from the USDA National Plant Germplasm System (NPGS) Malus collection in Geneva, NY over three seasons and identified valuable genetic resources for breeding and researching dihydrochalcones. From these resources, five apple hybrid populations were developed to determine the genetic basis of dihydrochalcone variation. Phloridzin, sieboldin, and trilobatin appear to follow segregation patterns for three independent genes and significant trait-marker associations were identified using genetic data from genotyping-by-sequencing. Dihydrochalcones are at much lower quantities in mature apple fruit compared with vegetative tissues. Within the fruit, phloridzin is more concentrated in the peel than in the flesh. We observed higher phloridzin content in the peel of russet apples compared to the peel of non- russet apples. Russet is a disorder affecting the development of the fruit cuticle, causing a smooth waxy surface to be replaced by a rough, russet colored layer. We compared leaf, and fruit peel and flesh samples from 108 accessions in the USDA-NPGS Malus collection and identified a strong correlation between percent russeting and phloridzin content the fruit peel, especially in sport families with variation for russeting. Though russeting can severely impact commercial value of apple cultivars, russeted fruit have a unique nutritional profile compared to non-russet cultivars. BIOGRAPHICAL SKETCH Benjamin Leo Gutierrez was born July 31, 1984 in Provo, UT, the youngest child of Ronald Phillip Gutierrez and Jean Crandall Gutierrez. His love of science began with teeth. Throughout his youth, he learned his father’s trade as dental technician, making dentures and other prosthetics in their basement laboratory, Timpanogos Dental Lab. From this, he developed a love for the diversity of form and function of teeth. To further his dental career, Benjamin enrolled at Utah Valley University, with a goal to pursue dentistry. The first in his family to attend college, he fell in love with science and learning. In 2005, Benjamin postponed his education to serve a two-year mission in Belgium and France for the Church of Jesus Christ of Latter-day Saints. Returning to Utah, Benjamin re-enrolled in college and took courses in genetics and plant physiology. Finding his love of plant science outweighed his love of teeth, he sought a new career as a scientist. He engaged in undergraduate research with Dr. Olga Ruiz Kopp, studying tissue culture of endangered Utah species, Astragalus holmgreniorum and pathogens of Pinus longaeva. Upon graduating from Utah Valley University in 2012 with BS in Biology and Botany, Benjamin applied for graduate studies at Cornell University to study plant breeding. There, he had the opportunity to work in partnership with Cornell’s apple breeder, Dr. Susan Brown, and Dr. Gan-Yuan Zhong of the USDA-ARS Plant Genetic Resources Unit at the New York State Agricultural Experiment Station (NYSAES). Throughout his graduate program, he had the unique opportunity to pursue advanced education and work for the USDA. In 2017, he was recognized for five years of service in federal government. The highlight of his experience was spending time in the apple repository, studying (and tasting) historic apple cultivars and wild species from around the world, and working with Dr. Brown, tasting the apples of the future. iii For my wife Emily, and our children Della, Leo, and Sullivan iv ACKNOWLEDGMENTS I am grateful to Dr. Susan Brown, for taking me on as a student and for all the time she spent searching the literature, reading drafts, making observations in the field and greenhouses, and offering much needed encouragement. I have learned from her passion for plant breeding, genetics, and the art of communication. With great confidence, she gave me freedom to expand my research project to fit my interests, and helped me to develop our apple populations. From her abounding humor, I learned to take my research seriously but not myself, and how to appreciate family and those who support me. As a good plant breeder, Dr. Brown sees value in things for what they could be, and not for what they are (including graduate students). I am grateful to Dr. Gan-Yuan Zhong, Research Leader, for the USDA-ARS Plant Genetic Resources Unit (PGRU). He provided important leadership both academically and professionally as my USDA supervisor and committee member. He was my voice to Area Office leaders in funding for research and ultimately a position within the unit through the USDA Pathways program. He advocated on my behalf of my value and contributions to the location. He was willing to talk about life, careers, and science, and was always very optimistic about my potential as a scientist in training. I would also like to thank my committee members Dr. Lailiang Cheng and Dr. Michael Gore. They provided key insights into my projects, technical experience, read early drafts, and allowed me to drop by their offices, often unannounced. I learned the principles of chromatography from Dr. Cheng, and to look beyond genetics, keeping physiological processes in mind. Dr. Gore taught me to think critically about statistics and quantitative genetics. I am grateful to have worked with Dr. Thomas Chao, Dr. Gennaro Fazio, and Dr. Heidi Schwaninger of PGRU. I learned much from them about Malus diversity, the value of v genetic resources, working with clonal crops, careful lab techniques, and how to think critically about my research. A special thanks to Dr. Jie Arro, a PGRU postdoc, who was always willing to talk about an idea for my projects, answer my questions about statistics, and how to love computer programming. Dr. Arro taught me how to run the GBS pipeline, and to think critically about data analysis. I also thank the ‘A-Team’, Bill Srmack, John Keeton, Bob Martens, and Julian Koob of PGRU. Working under Bill Srmack’s leadership, they keep the collections alive and in excellent condition. Truly, it is their work that people come from all over the world to see. They were always quick with a joke, and took time to teach me about horticulture and management of the clonal repository, even when I got vehicles stuck in the mud. Though he’s moved on from our unit, I am grateful to Andy Humiston, who taught me to run and maintain our HPLC. We spent a lot of hours together learning the art of chromatography. I am grateful to Julian Koob for his help in preparing and extracting samples for HPLC. I am grateful to Kevin Maloney, whose good humor and vast knowledge of apples improved the quality of my research and my life. Without Kevin and Matt Garlick’s technical assistance, I would not have been able to pollinate, germinate, and plant the 1,000+ seedlings that were irreplaceable to my research. I am also grateful to former lab member, Dr. Benjamin Orcheski, who made crosses for another project that turned out to be invaluable for mine, and suggested we look at russeted apples. I also thank Dr. Rose Nichols, Tiffany Fisk, and Martin Hooker with the USDA who keep everything running smoothly and are the heart of the USDA-ARS Geneva location. This project was funded by the USDA-ARS Plant Genetics Resources Unit, Geneva NY. I am grateful to the Plant Breeding and Genetics section, the New York State Agricultural Experiment vi Station (NYSAES), and the United States Department of Agriculture for their support. I also thank the staff, graduate students, and faculty in Geneva, NY who make NYSAES a wonderful place to work. I am grateful to the Arnold Arboretum of Harvard University for providing budwood of Malus trilobata 127-2009. Finally, I am grateful to my wife Emily Ann Gutierrez, and to our children Della Jean, Leo Jay, and Sullivan Ronald. They were always supportive, but excited when I’d come home to play. My wife spent many hours discussing my project with me, and learned to pronounce botanical names and chemical compounds. My children gave me a lot of exercise. Collecting leaves, and hunting for rocks and bugs with them retaught me how to see the natural world, and enjoy it with childlike wonder. My family kept life in perspective and helped me stay focused on what matters most. vii TABLE OF CONTENTS BIOGRAPHICAL SKETCH iii DEDICATION iv ACKNOWLEDGMENTS